| Background:Liver injury refers to chronic liver damage caused by a variety of causes,including viral hepatitis,alcoholic liver disease and drug-induced liver damage.The changes in the internal and external environment of the body exceed the adaptive capacity of cells and tissues,resulting in abnormal changes in material metabolism,morphological structure and function of damaged cells and cell stroma,which are called liver injury.The injury is manifested in hepatocyte degeneration,necrosis and abnormal liver function,while the antiinjury response includes local and systemic inflammatory reaction,hepatocyte regeneration and repair.The basic mechanisms include pathogenic microbial infection,immune liver injury,chemical liver injury,heat stress liver injury and so on.Immune factor-mediated liver injury is one of the main types.Clinically,liver injury is characterized by chronic development and repeated procrastination.After the initial inflammatory reaction is aggravated,it will lead to liver cirrhosis,liver cancer and other complications for a long time.Modern medicine has certain pertinence in the treatment of liver injury according to the characteristics of pathogenesis,for example,for CHB(chronic hepatitis B),nucleosides and interferon are mainly used against HBV(hepatitis B virus),while liver cirrhosis,alcoholic liver disease and drug-induced liver disease are mainly treated by protecting liver and lowering enzymes and anti-liver fibrosis.Its purpose is to alleviate the necrosis and apoptosis of hepatocytes and the further damage of liver function,and to prevent the progressive aggravation of liver fibrosis,liver cirrhosis and the pathological changes of secondary liver cancer.Modern medicine has a positive effect on liver-protecting and enzyme-lowering treatment and etiological treatment of liver injury,especially the anti-HBV effect of nucleoside analogues and interferon can alleviate the condition of a large number of patients with CHB and liver cirrhosis.However,more and more studies have shown that the liver protective treatment of chronic liver function damage is lack of specificity,and the control of the progression from hepatic fibrosis to liver cirrhosis is not very ideal.Nucleosides should be used for a long time,and the side effects are obvious.It is easy to rebound after drug withdrawal,and even liver necrosis occurs.A large number of studies have shown that traditional Chinese medicine shows good effects and potential advantages in the clinical research field of treating liver injury.Jiangan Powder(JGP)is an empirical prescription in the treatment of chronic liver disease in our hospital for many years,and its clinical effect is remarkable.It has the TCM efficacy of soothing the liver and invigorating the spleen,invigorating the vital energy and dispelling evil,and reconciling qi and blood.The prescription is based on the TCM treatment view of "the same tone of liver and spleen" and "corresponding prescription and syndrome" of chronic liver disease.The previous clinical and experimental studies show its objective curative effect.In order to further evaluate the curative effect of the prescription,this paper discusses the relevant theoretical basis,clinical function and biological mechanism of the curative effect,in order to reveal the scientific connotation of the therapeutic effect of the prescription and provide a scientific basis for further clinical application.Part Ⅰ:Theoretical researchThe summary of theoretical literature is as follows:(1)Epidemiological studies show that the incidence of chronic liver injury is high in China,with a large base and high incidence.The main pathogenesis structures are chronic viral hepatitis(B),autoimmune liver disease,druginduced hepatitis,alcoholic liver disease and liver cirrhosis,of which viral hepatitis is the most common.(2)the pathogenesis of chronic liver injury is different,and immune-mediated inflammation is the main basic mechanism,and the disturbance of intestinal flora has an effect on the pathogenesis of liver injury.the immune injury of liver tissue is the consequence of the synergistic effect of liver tissue specific immunity and innate immunity,in which non-specific inflammatory cells such as macrophages are the main effector cells of liver tissue injury.(3)Modern medicine is objective and effective in protecting liver and lowering enzyme,antivirus and other related therapeutic drugs for chronic liver injury,but it can not completely effectively block the process of chronic liver injury and liver fibrosis.and there are many side effects and the malpractice of rebound after drug withdrawal.(4)traditional Chinese medicine believes that the pathogenesis of liver injury is mainly related to pathological factors and pathological products such as dampness,heat,blood stasis and toxin,and its location is mainly in the liver,mainly involving spleen and kidney.The disease belongs to deficiency in origin and excess in superficiality,and deficiency and excess are mixed.Liver depression and spleen deficiency is the key pathogenesis and main syndrome type of liver injury,and soothing the liver and invigorating the spleen is its treatment method.(5)many studies have shown that protecting liver and lowering enzymes,relieving inflammatory reaction,improving symptoms,regulating immune microenvironment and anti-liver fibrosis are the advantages of traditional Chinese medicine in the treatment of liver injury.(6)as a clinical empirical prescription for the treatment of chronic liver disease,dialectical legislation has its own theoretical basis,which has the effect of soothing the liver and invigorating the spleen,invigorating the spleen,invigorating the vital energy and dispelling evil,and reconciling qi and blood.(7)based on the action mechanism of "intestinal liver axis",regulating the intestinal flora and its metabolites of the host with liver injury can effectively alleviate the occurrence and development of liver injury.(8)the activation of STAT3 signal pathway is related to the occurrence and development of liver injury.Regulating the expression of STAT3 signal pathway related proteins can effectively interfere with the expression of related inflammatory factors and alleviate liver injury.The theoretical literature research briefly summarized the pathogenesis,pathological mechanism and treatment of chronic liver injury,which laid a theoretical foundation for exploring the scientific connotation of the therapeutic effect of JGP on chronic liver injury.Part Ⅱ:Network Pharmacology and Molecular dockingObjectiveThe targets and related pathways of JGP in the treatment of liver injury were analyzed and verified by network pharmacology and molecular docking methods,and the related mechanisms were discussed.Methods(1)Search the effective chemical components and corresponding targets of JGP in TCMSP,and obtain the target information;(2)use DisGeNET,GeneCard and OMIM database to mine and obtain liver injury targets;(3)screen the relevant targets of active components in JGP and liver injury targets by Venny online software,that is,the potential targets of active components in JGP in the treatment of liver injury.(4)Gene Symbol of drug and disease intersection target was introduced into STRING database platform,and protein interaction(PPI)analysis was carried out.(5)significantly enriched molecular signal pathway and metabolic pathway were obtained by GO analysis and KEGG analysis to explore the signal pathway and biological process of JGP in the treatment of liver injury.(6)molecular docking method was used to further verify the interaction between the predicted key active components and targets.Results(1)468 active ingredient targets and 1014 liver injury therapeutic targets in JGP were predicted by preliminary screening,and a total of 158 potential targets were obtained after matching.(2)Quercetin der,naringenin,Glyasperin B,Didymin,sophoridine,matrine,(6S)-6(hydroxymethyl)-1 and(6S)-6-(hydroxymethyl)-1 were selected from the active components of JGP.There were more matching targets of naphtho,benzofuran-10,11-dione and Chrysoeriol,which may be the key effective components of JGP in the treatment of liver injury,and most of them were alkaloids and flavonoids.Related studies show that these targets have the effects of anti-inflammation and antioxidation,regulating lipid metabolism and alleviating hepatocyte injury.(3)the targets with the highest values obtained by PPI analysis include TNF,AKT,STAT3,CASP3 and CXCL8 may be the key targets of JGP.(4)the results of GO and KEGG enrichment analysis showed that JGP could alleviate the injury of hepatocytes caused by chemical stimulation and oxidative stress through signal transduction,regulation of receptor protein phosphorylation and regulation of phosphokinase activity,promote hepatocyte proliferation,and promote liver regeneration,alleviate hepatic fibrosis,reduce steatosis and inflammatory injury through HIF-1,AKT and RAS pathways.(5)the core protein obtained by PPI analysis was docked with the corresponding active components.The results showed that all the small molecules could bind stably with the ligand and the binding of STAT3,HSP90AA1 and MMP9 was the most stable.ConclusionA variety of active compounds in JGP play a coordinated role,multi-channel and multitarget to alleviate liver immune inflammation and injury,inhibit liver fibrosis,regulate immune balance and so on.In this research,through the methods of network pharmacology and molecular docking,we screened the potentially active compounds and targets of JGP in the treatment of liver injury,and verified the possible mechanism of pharmacological action based on STAT3 and other related targets.Part Ⅲ:Research on effective components of JGPObjectiveThe effective components of JGP and the effective substances of metabolism in vivo were studied and analyzed by UHPLC-Q-TOF-MS technology,and the material basis of its therapeutic effect was discussed.MethodsThe water extract of compound JGP was routinely prepared,and the SD male rats were given intragastric administration according to 10.71 g/kg·d for 7 days.The drug-containing serum samples were prepared.According to the effective catabolism products suggested by the results of network pharmacology,the metabolites in JGP water extract and drug-containing serum were analyzed by UHPLC-Q-TOF-MS technique and chemical composition characterization analysis.ResultsTwo main types of compounds were found in JGP serum:alkaloids and flavonoids.Alkaloids are matrine,sophoridine,sophorine,laminine,isophocarpine,etc.Flavonoids,mainly naringenin,Glyasperin D,Glyasperin M,glycyrrhizinol A,zelanesin,kaempferol-7 color Orhamnoside,etc.ConclusionThe therapeutic effect of JGP on liver injury is related to the effective components of alkaloids and flavonoids contained in the prescription.The above compounds may be the main material basis for its curative effect.Part Ⅳ:Experimental researchObjective(1)To explore the inhibitory effect of JGP on the mouse model of immune liver injury induced by BCG and LPS,so as to explain the preventive and therapeutic effect of JGP on liver injury.(2)to explore the preventive and therapeutic mechanism of JGP on immune liver injury by observing the effects of JGP on intestinal flora structure,bacterial changes and metabolites in mice with liver injury.(3)to study the possible molecular mechanism of JGP in the prevention and treatment of liver injury by observing the regulatory effect of JGP on the expression of STAT3,p-STAT3 protein and PCNA antigen in mice with liver injury.Methods(1)Grouping:25 SPF male Balb/c mice were divided into normal group(group A),model control group(BCG+LPS group)(group B),low dose group of JGP(group C),middle dose group(group D)and high dose group(group E).(2)intervention:groups An and B were given 0.2mL saline intragastrically from the first day.The mice in the other groups were intragastrically perfused with the corresponding drug 0.2mL for 12 days.At the same time,except for group A,the mice in the other groups were injected with 0.2mL BCG solution through the tail vein on the first day,0.2mL LPS saline solution was injected into the tail vein 12 days after sensitization,and the mice in group A were injected with the same volume of normal saline.(3)to observe the changes of body weight,spleen and thymus organ index after intervention.(4)the abundance,diversity,structure,metabolite composition and metabolic pathway of intestinal contents of mice were observed and analyzed by 16s rDNA and LC-MS non-target metabonomics,(5)the protein expression of STAT3 and p-STAT3 was detected by Western blot,the expression of PCNA was detected by IHC,the level of ALT and AST in serum was detected by biochemical method,and the content of IFN-γ,IL-6 and IL-22 in serum was detected by Elisa.Results1.Changes of body weight,spleen and thymus organ index in each group:the results showed that the thymus index in the model group was significantly higher than that in the normal group,and the thymus index in each group was significantly lower than that in the model group after JGP intervention,suggesting that JGP has a certain immunosuppressive effect on the thymus of mice with immune liver injury.2.Effect on intestinal microflora:the results of 16s rRNA sequencing showed that the diversity of intestinal microflora in mice with immune liver injury was significantly increased,and the diversity and richness of intestinal microflora were significantly improved in the middle and low dose groups of JGP,but the diversity and richness of intestinal microflora decreased in high dose JGP treatment.PCoA analysis showed that there were significant differences in the structure of intestinal flora between the normal group and the model group,while the middle and high dose groups of JGP showed similarities with the normal group,suggesting that the immune liver injury caused by BCG+LPS changed the overall structure of intestinal microflora.JGP can regulate and maintain the intestinal tract in immune dynamic balance under the change of microflora.At the generic level,Alloprevotella,Burkholderia-Caballeronia-Paraburkholderia,Muribaculum,Streptococcus and Stenotrophomonas are considered to be the main targets for JGP to play a regulatory role.3.Metabonomic analysis:studies have shown that JGP can correct the disorder of biotin metabolism and steroid biosynthesis.The biosynthesis of arginine may play a role in the hepatoprotective effect of JGP.Compared with the model group,the effect of arginine biosynthesis in the high dose group of JGP is significant.JGP can reverse the contents of Allylestrenol,Eplerenone,PE,SM d27purl,Soyasapogenol C,Chrysin and Soyasaponin I in intestinal contents of mice with immune liver injury.It is considered to be the metabolic target of JGP.The regulation of Allylestreno and Chrysin and related intestinal flora metabolites may be the potential mechanism of JGP in hepatoprotective effect.4.Regulatory effect on related pathway proteins and cytokines:the results of WB experiment showed that the expression of STAT3 protein in the model group was significantly higher than that in the normal group.JGP treatment could reduce the expression of p-STAT3,but did not affect the level of STAT3.It is suggested that JGP may reduce cellular inflammatory response by inhibiting the phosphorylation activation of STAT3 signal pathway.In addition,the expression of NF-κB protein in the model group was significantly higher than that in the normal group,and the expression of JGP in each group was lower than that in the model group.The results of PCNA immunohistochemistry showed that the percentage of PCNA positive cells in the middle and high dose of JGP was higher than that in the model group,suggesting that JGP could increase the expression of PCNA.The expression of IFN-γ,IL-6 and IL-22 in the model group was significantly higher than that in the normal group(P<0.001),and decreased significantly after the intervention of JGP(P<0.001),suggesting that JGP can inhibit the release of inflammatory factors produced by the liver,thus alleviating the inflammatory response of the liver.5.Effect on liver function:the results of this research showed that the serum contents of ALT and AST in the model group were significantly higher than those in the normal group,and significantly decreased after intervention in all dose groups of JGP(P<0.05),indicating that JGP has a protective effect on liver function in mice with immune liver injury.ConclusionJGP can(1)improve the body weight,spleen and thymus organ index of mice with liver injury,(2)affect the diversity and composition of intestinal flora and regulate their metabolites by targeting intestinal flora,(3)down-regulate the expression of STAT3/p-STAT3 and NF-κB,increase the expression of PCNA,reduce the levels of serum ALT and AST,regulate cytokines such as IFN-γ,IL-6 and IL-22,so as to reduce inflammation.(4)improve liver function and promote hepatocyte proliferation,so as to improve liver injury.The above results reveal the therapeutic mechanism of JGP on liver injury to some extent.Part Ⅴ:Clinical researchObjectiveTo observe the clinical efficacy of JGP in the treatment of chronic liver disease-CHB,and to comprehensively evaluate the advantages of entecavir combined with JGP in protecting liver and lowering enzymes,reducing inflammatory reaction and improving symptoms of CHB,so as to provide objective basis for clinical application.MethodsA total of 40 patients with CHB liver injury diagnosed as liver depression and spleen deficiency in Hai’an Traditional Chinese Medicine Hospital Gastroenterology Clinic from January 2021 to December 2021 were randomly divided into observation group(JGP combined with entecavir)and control group(entecavir).The treatment was observed for 8 weeks.To observe and compare the changes of liver function(ALT,AST,Tbil,ALB),HBV-DNA,inflammatory factor indexes(TNF-α,IFN-γ,IL-6,IL-22),TCM symptom score and TCM syndrome curative effect between the two groups before and after treatment.Results1.Changes of TCM symptom scores:after 8 weeks of treatment,the total symptom scores of the two groups were significantly improved as compared with those before treatament(P<0.05),and the improvement rate of main symptom scores of the observation group was 75.4%,which was significantly higher than that of 49.1%of the control group(P<0.05).So that loose symptoms can be improved.The secondary symptoms in the observation group were improved by 82.5%compared with those before treatment,which was also better than that in the control group(56.6%).The improvement rate of secondary symptoms such as bitter or sticky mouth,abdominal pain,intestinal ringing and do not drink when thirsty was better than that in the control group(P<0.05).2.Therapeutic effect of TCM syndrome:the results showed that the effective rate of the observation group was 100%and that of the control group was 90%,showing the curative effect advantage of the observation group(P<0.001).3.Laboratory test:the changes of liver function:after treatment,the liver function of the two groups was improved,and the ALT of the observation group decreased significantly,which was better than that of the control group(P<0.05).The AST,Tbil and ALB of the two groups were also improved in varying degrees compared with those before treatment,although there was no statistical significance between the two groups(P>0.05).4.HBV-DNA:after treatment,the levels of HBV-DNA in both groups were significantly lower than those before treatment(P<0.05),but there was no significant difference between the two groups(P>0.05).5.Changes of cytokines:after treatment,TNF-α,IL-6 and IL-22 in the two groups were significantly lower than those before treatment(P<0.05),and the observation group was significantly better than the control group(P<0.05).ConclusionAfter entecavir combined with JGP,the clinical efficacy of CHB was significantly increased,and it showed different advantages or tendencies in symptomatic changes,liver function changes,inhibition of inflammatory factor expression and inhibition of HBV-DNA replication,especially in improving symptoms,TCM syndrome and reducing ALT.We believe that there is a synergistic effect of nucleoside analogue entecavir combined with JGP in the clinical treatment of CHB. |
PDF Full Text Request